In order to investigate how energy expenditure changes with over and underfeeding the following studies are underway. In one study, after careful calibration of weight maintenance EE, individuals undergo a series of measurements of 24 hour EE in a respiratory chamber in which they are fasting or overfed (by 200% of weight maintenance needs) a series of diets that vary in macronutrient content. This is to further investigate whether low or high protein diets may improve the detection of recruitment of adaptive thermogenesis. In addition, behavioral, metabolic and hormonal tests are performed to examine associated characteristics and to investigate the mechanism of the changes in EE. These individuals are followed up long term (up to 7 years) to look at factors which predict weight change. We have demonstrated that the change in energy expenditure with fasting and with overfeeding is reproducible. We have had 77 participants complete the core portion of this study. We have found a negative association between the percent change in EE with overfeeding with the percent change in EE with fasting indicating the presence of thrifty versus spendthrift phenotypes. We also found that high carbohydrate and high protein overfeeding increased EE the most, while low protein overfeeding increased EE the least. At 6 months, we found that individuals with greater decrease in EE with fasting and less increase in EE with low protein overfeeding gained more weight. These data again indicate the presence of more thrifty phenotypes. However we also found that higher EE change in response to high carbohydrate diet predicted greater weight gain and that those with higher EE after high carbohydrate overfeeding were also hungrier. Thus, high carbohydrate overfeeding may stimulate energy sensing mechanisms that drive food intake. We have been investigating the mechanisms underlying these energy expenditure changes to fasting and overfeeding. We found that core body temperature correlated with changes in EE in response to fasting, such that individuals with lower core body temperature have a greater decrease in EE with fasting. We have also found that urinary epinephrine concentrations are higher in those with more thrifty phenotypes. We are continuing long term follow-up of those with these energy expenditure measurements and investigating the role of the sympathetic nervous system, gastrointestinal and adipocyte hormones as possible mediators of these EE changes and or weight change. Based on the difference in energy expenditure measured during fasting and overfeeding, the thermic effect of food (TEF) can be calculated. We have found that TEF is inversely associated with adiposity. Thus TEF may predispose to weight gain or be an adaptation to increased bodyweight. As increased adiposity may insulate against trans-abdominal heat loss which may increase TEF, we are investigating the effect of central insulation on the EE and TEF changes associated with overfeeding. Fourteen of the needed 20 participants have completed this part of the study. Because of the recent discovery of the presence of brown fat in humans and its possible role in thermogenesis, we performed positron emission scans with labeled glucose. As brown fat is activated by cold temperatures, we have established that we can visualize brown fat after 2 hours of exposure to 16 degrees Celsius. We then currently investigated whether individuals with visualized brown fat after cold exposure, have visualized brown fat after overfeeding. Following demonstration of visible brown fat after cold exposure individuals were overfed by 200% of their energy needs using a high fat normal protein diet while in our metabolic chamber. The next morning they underwent a PET-CTscan; this was performed in some individuals prior to breakfast (approximately 12 hours after their last overfeeding meals) and in some individuals following a similar overfeeding breakfast (approximately 4 hours after their last meal). We found no evidence of activation of brown fat with overfeeding following a high fat overfeeding, indicating that brown fat does not mediate the increased energy expenditure associated with overfeeding. Based on evidence of brown fat activation with high carbohydrate overfeeding, we have used the same overfeeding paradigm with our high carbohydrate overfeeding diet followed by PET-CT scans. In preliminary analysis, we have found evidence of brown activation with this diet. We are confirming these results and are currently developing a new study to confirm and investigate the role of overfeeding in brown fat activation. In a related study, after measurement of EE with over and underfeeding, and also after undergoing a series of metabolic and behavioral testing (including biopsies of muscle and fat), individuals are admitted for 6 weeks of an inpatient dietary protocol involving underfeeding (for overweight and obese individuals) or overfeeding (for lean, obesity resistant individuals). During the inpatient study, all aspects of food intake, energy expenditure, and energy loss are carefully measured to determine if differences in weight gain or loss can be attributed to recruitment of adaptive thermogenesis or other factors. We have currently completed 12 individuals who have undergone the weight loss study. In these individuals, we found that the calculated energy deficit (based on measured loss of fat mass and fat free mass) did not significantly differ from the deficit as calculated based on energy intake, measured energy expenditure, and stool and urine losses. We did find that less decrease in 24hEE with fasting was associated with greater weight loss in this controlled inpatient study. Participants with the greatest accumulated measured energy deficit lost the most weight, and greater measured energy deficit was also associated with less 24h EE decrease with fasting. Consistent with our natural history study described above, these results indicate the presence of spendthrift versus thrifty phenotypes even in obese individuals undergoing weight loss, and accounts in part for the variability in dietary weight loss. We are currently investigating the pathways that might explain this variability in EE response, and have performed RNA sequencing on collected muscle and adipose tissue in these individuals prior to and following weight loss. Recruitment for both studies is ongoing.